Remote ischemic conditioning (RIC) confers protection on major organs from hypoxic/ischemic injuries; however, its impacts on attention network function and blood oxygen levels in unacclimatized adults exposed to high altitudes have yet to be elucidated. In this study, we recruited 120 healthy male volunteers, of which one was exposed to high altitude and the other was exposed to low altitude. The two cohorts were further divided into RIC and sham control groups. The attentional network test (ANT) was performed to evaluate cognitive function before and after RIC treatment. Other outcomes such as heart rate, blood pressure, blood oxygen saturation, cerebral tissue oxygenation index (CTOI), and cerebrovascular hemodynamic indices were also evaluated. Prior to RIC treatment, there were no significant differences in orienting or executive function between the treatment and control arms of either cohort. Alerting function was significantly lower in the high-altitude cohort than in the low-altitude cohort. There were significant reductions in both blood oxygen and CTOI in the high-altitude cohort relative to the low-altitude cohort, while the pulse index (PI) of the former cohort was significantly increased. After RIC treatment, there was a significant difference in alerting function between the high-altitude RIC group and its associated control. The CTOI of the treatment group increased from 60.39±3.40% to 62.78±4.40%, and blood oxygenation also improved. Furthermore, this group showed a significant reduction in its PI. Exposure to high-altitude environments had a significant impact on alerting function, blood oxygen, CTOI, and PI. RIC ameliorated changes in attentional function, as well as blood oxygen and CTOI, suggesting that it potentially alters cerebrovascular compliance upon exposure to high altitude. High altitude has been shown to cause a series of pathophysiological changes via hypoxia, low atmospheric pressure, cold, and other environmental factors , which mRemote ischemic conditioning (RIC) is a systemic strategy in which several cycles of ischemia followed by reperfusion in the limbs confer protection on distant vital organs , 14. RICRecently, some studies have demonstrated that the attentional network can be disturbed by brief exposure to high altitude -21. In aWe recruited two cohorts of subjects. The first cohort included 60 young healthy male volunteers who were newcomers to the region and had arrived at an altitude of 3,700 meters on the Tibetan Plateau 3 days before entering the study. The other cohort included 60 young healthy male volunteers who remained at an altitude of 42 meters on the plains and had never lived in a high-altitude region . These two cohorts of subjects were randomly assigned to RIC or sham group (n=30 in each group). Other inclusion criteria for this study included the following: (1) age between 18 and 30 years old; (2) right-hand dominance, have normal or corrected-to-normal vision, absence of intellectual disability, and no history of neurological/psychiatric illness or hemorrhagic diseases; (3) no history of intravascular thrombosis in either of the upper limbs.All subjects underwent RIC or sham-RIC treatment for one week, beginning on the third day of exposure . The RIC2) with a portable oxygen saturation apparatus designed for hospital wards , and we measured the cerebral tissue oxygen index (CTOI) with a cerebral oxygen monitor .The heart rate and blood pressure of each subject was acquired before and after RIC treatment. We monitored blood oxygen saturation , as described by Fan  in 2002 , was admAll cerebral hemodynamic data were collected using a transcranial doppler  according to applicable specifications and technical standards before and after one week of treatment. The peak systolic blood flow velocity (PSV), end-diastolic blood flow velocity (EDV), and mean flow velocity (MFV) of the subjects’ bilateral middle cerebral artery (MCA) were recorded, and the pulse index (PI) was calculated.For continuous data, the mean ± standard deviation (SD) was used to summarize the data; independent-samples t-tests or Mann-Whitney U tests were performed to detect differences between groups. For binary data, frequency or percentage was used to summarize data, and between-group comparisons were performed via the chi-squared test or Fisher’s exact test as appropriate.All data were analyzed using SPSS 19.0 (IBM Statistics) with a significance level of p<0.05 (two sides).The demographic and clinical characteristics of the subjects in the four groups are summarized in No differences were observed between the high-altitude and low-altitude groups in terms of heart rate or blood pressure. In the high-altitude cohort, the blood oxygen and CTOI of the sham-RIC group were 90.93±2.07 and 59.13±2.53, respectively, and those of the RIC group were 91.13±2.07 and 58.14±2.79; those of the low-altitude cohort were 94.23±2.53 and 65.95±3.62 in the sham-RIC group and 93.67±2.13 and 65.15±3.64 in the RIC group .Both parameters were significantly different between the two cohorts (P<0.001). The alerting function of the high-altitude cohort was 28.53±19.62 and 27.87±12.85 in the sham-RIC group and RIC group, respectively; these values were significantly lower than those of the low-altitude group , and the blood oxygenation was also higher with RIC than with sham treatment . Together, these results indicate that blood oxygen saturation and CTOI were significantly improved by RIC treatment. There were no significant differences in orienting  or executive function . However, after RIC or sham treatment of the high-altitude cohort, the alerting score of the sham-RIC group was 26.96±18.18, while that of the RIC group was significantly higher at 48.80±24.32 P<0.001; .No differences were observed between the high-altitude and low-altitude cohorts in terms of the PSV, EDV or MFV in the bilateral MCA of the volunteers. The PI, however, was significantly (P<0.01) higher bilaterally in the high-altitude group than in the low-altitude group  was 0.93±0.09% and 0.92±0.12%, respectively, in the sham-RIC and RIC groups; PI of the right MCA (RMCA) was 0.93±0.11% and 0.93±0.13%, respectively, in those two groups; low-altitude cohort: PI of the LMCA was 0.83±0.10% and 0.82±0.09%, respectively, in the sham-RIC and RIC groups; PI of the RMCA was 0.81±0.13% and 0.80±0.08%, respectively, in those two groups; In this study, we found that, compared with the low-altitude cohort, the high-altitude cohort had diminished alerting function, while their orienting and executive function remained unchanged. In addition, this reduction in alerting function was significantly improved after one week of RIC treatment in the high-altitude cohort population, suggesting that RIC treatment is a promising strategy to cure cognitive impairment during high-altitude exposure.The human attention network model proposed by Posner and Petersen  postulatet al. [Our study showed that blood oxygen saturation was lower in the high-altitude group than in the low-altitude group. According to the ANT, the alerting function of the high-altitude subjects was reduced compared to that of the low-altitude subjects. Alerting is the ability to prepare for a sensory signal, allowing one to achieve and maintain a highly sensitive state of vigilance in which one is ready for any incoming stimuli. Godfrey et al.  found thRIC is a phenomenon in which a brief period of ischemia in one organ or tissue protects another organ or tissue against sustained ischemia-reperfusion injury , 26. PreThe current study has some limitations. The study recruited only a small sample size, and the follow-up period was short; these limitations may have introduced some biases influencing the results. At the same time, although this study found improvement in attention network function after RIC treatment, the exact mechanism through which RIC exerts that effect was not investigated in depth. Finally, since only male volunteers were recruited in this study, the consequent sex bias may impact the generalizability of these results.In conclusion, short-term exposure to high altitudes produced a significant impact on alerting function, and RIC treatment is a useful strategy to mitigate the reduction in alerting function in high-altitude or other hypoxic environments. Further studies are necessary to further confirm these results and to explore the underlying mechanisms.


Renal cell carcinoma (RCC), which was one of the most common malignant tumors in urinary system, had gradually increased incidence and mortality in recent years. Although significant advances had been made in molecular and biology research on the pathogenesis of RCC, effective treatments and prognostic indicators were still lacking. In order to predict the prognosis of RCC better, we identified 17 genes that were associated with the overall survival (OS) of RCC patients from The Cancer Genome Atlas (TCGA) dataset and a 17-gene signature was developed. Through SurvExpress, we analyzed the expression differences of the 17 genes and their correlation with the survival of RCC patients in five datasets , and then evaluated the survival prognostic significance of the 17-gene signature for RCC. Our results showed that the 17-gene signature had a predictive prognostic value not only in single pathologic RCC, but also in multiple pathologic types of RCC. In conclusion, the 17-gene signature model was related to the survival of RCC patients and could help predict the prognosis with significant clinical implications. Renal cell carcinoma (RCC), the main form of kidney cancer, was the second most common cancer in the urological system and accounted for approximately 3% of malignant neoplasms worldwide . It inclAlthough surgical tumor resection was an effective treatment for RCC at present –12, radiIn order to solve this unmet need, a prognostic 17-gene signature model was developed. We hypothesized that this 17-gene signature may reflect the risk level of adverse clinical outcomes in RCC, which may be useful to correctly predict the overall survival (OS) of RCC and aid the clinicians in treatment planning.http://bioinformatica.mty.itesm.mx:8080/Biomatec/SurvivaX.jsp). SurvExpress was a comprehensive gene expression analysis tool that was based on a number of databases. It can provide risk assessment and survival analysis in many cancer datasets [p < 0.001). After that, the 3761 genes were sorted by p value and the top 99 genes were combined. In this combination, genes with a significant difference in expression levels were picked out to form a 17-gene signature , and then evaluated the survival prognostic significance of the 17-gene signature for RCC . The prop < 0.05 was considered to be statistically significant.To evaluate the prognostic value of the 17-gene signature, Kaplan-Meier estimator was used to plot survival curves and the log-rank test was performed to compare the differences between the two groups , 27. Kapp < 0.001)  .Sort PI from low to high and select the optimal cut-off value. The optimal cut-off values of PI in the five databases  were 11.13, 14.67, 16.5, 16.81, and 0.355 . Then, tn = 272) was significantly better than high-risk group patients (n = 196) (HR (95%CI) = 4.05 (2.85-5.74), p < 0.001) (p < 0.001) (n = 240 in the low-risk group and n = 38 in the high-risk group) (HR (95%CI) = 9.58 (4.97-18.45), p < 0.001) (p < 0.001) (n = 115) and the high-risk group (n = 62) (HR (95%CI) = 2.27 (1.49-3.47), p < 0.001), indicating that the 17-gene signature can also be used as a prognostic indicator in the ZHAO database  . KIRC da< 0.001) . The res< 0.001) . KIPAN i< 0.001) . ZHAO dadatabase . In concIn order to determine the accuracy of the 17-gene signature in predicting the prognosis of RCC, we conducted the true positives and false positives analysis by SurvExpress . The recRCC was the third most common malignancy in the urogenital system, which represented about 2% to 3% of cancers in adults . The genα) [RCC was classified as an “immunogenic” tumor based on the following characteristics: spontaneous regression of the tumor, high levels of T cell infiltration in the tumor, and reactivity to immunotherapy such as interleukin-2 (IL-2) and interferon alpha (IFN-α) . Howeverα) . Eventuaα) . Therefop < 0.001). Then, a 17-gene signature model was developed and its correlation with the survival of RCC patients in the five datasets was analyzed, respectively. The patients were divided into low-risk and high-risk groups according to the optimal cut-off value. Survival differences between the predicted low-risk and high-risk groups were evaluated with Kaplan-Meier survival curves. The results presented that the prognosis of patients with low risk was all significantly better than that of high-risk group patients in the five databases (p < 0.001), suggesting that the 17-gene signature had a predictive prognostic value not only in single pathologic RCC but also in multiple pathologic types of RCC.In the present study, we first identified 17 genes associated with OS of RCC patients from the TCGA dataset. Through SurvExpress, we analyzed the expression differences of the 17 genes in RCC patients in the TCGA dataset. Our analysis showed that the 17 genes had significant prognostic differences in the TCGA database (p < 0.001), suggesting that the 17-gene signature had certain accuracy in predicting the prognosis of RCC.Finally, in order to determine the accuracy of the 17-gene signature in predicting the prognosis of RCC, the receiver operating characteristic curve (ROC) for predicting 5-year survival was obtained according to PI. The value of AUC was the size of the area under the ROC curve. Typically, the AUC value was between 0.5 and 1.0, and larger AUC represented better performance , 31. OurIn summary, our study demonstrated a survival prognostic significance of a 17-gene signature for RCC. This may be a potential prognostic tool to improve the adverse clinical outcomes of RCC patients currently. Further prospective studies were needed to determine whether the 17-gene signature can be used clinically to benefit RCC patients.


In recent years, pepino mosaic virus (PepMV) has rapidly evolved from an emerging virus to an endemic pathogen, as it causes significant loses to tomato crops worldwide. At present, the main control strategy for prevention of PepMV disease in tomato production remains based on strict hygiene measures. To prevent damage caused by PepMV, cross-protection is used in some countries. Reliable characterisation, detection and quantification of the pathogen are vital for disease control. At present, reverse-transcription real-time quantitative polymerase chain reaction (RT-qPCR) is generally used for this purpose. However, quantitative use of RT-qPCR is linked to standardised reference materials, which are not available for PepMV. In addition, many factors can influence RT-qPCR efficiencies and lead to lower accuracy of the quantification. In this study, well-characterised PepMV-genotype-specific RT-qPCR assays were transferred to two digital PCR (dPCR) platforms. dPCR-based assays allow absolute quantification without the need for standard curves, and due to the binary nature of the reaction, dPCR also overcomes many of the other drawbacks of RT-qPCR. We have shown that these newly developed and validated PepMV-genotype-specific dPCR assays are suitable candidates for higher-order methods for quantification of PepMV RNA, as they show lower measurement variability, with sensitivity and specificity comparable to RT-qPCR. Potexvirus and has virions that are non-enveloped flexuous rods of 508 nm in length [Solanum muricatum) [Lycopersicon esculentum) in 1999 in greenhouses in The Netherlands [Pepino mosaic virus (PepMV) belongs to the genus n length . The Pepn length . It was ricatum) , and washerlands . Shortlyherlands . PepMV cherlands ,7. The dherlands ,8.Pepino mosaic virus isolates show considerable genomic diversity, which appears to be linked to the geographic origins, specific hosts and/or the high mutation rates of RNA viruses. These differences appear to increase adaptation of PepMV to new environments . There aPepino mosaic virus is highly infectious, and it is readily transmitted through mechanical means. It can be spread easily by plant-to-plant contact, and during routine cultivation procedures, while seed transmission might have a role in its long-distance spread . Water hThe PVM-01 vaccine was designed on the principle of cross-protection, which uses a mild isolate as a protective agent against more aggressive strains. Such cross-protection depends strongly on the genotype, and it is effective only under well-defined conditions . For theReverse-transcription real-time quantitative polymerase chain reaction (RT-qPCR) is at present still the method of choice for quantitative detection of the PepMV genotypes . This quIn contrast to real-time quantitative PCR (qPCR), digital PCR (dPCR) can provide absolute quantification of target sequences without reliance on standard curves. By partitioning the samples into thousands of reactions before amplification, the copy number in the initial sample can be determined based on the number of positive and negative partitions after end-point PCR amplification, using Poisson’s statistics . As wellToday there are several dPCR platforms available, which can be divided into two general groups: emulsion-based or droplet dPCR; and chip-based dPCR . For theThe aim of the present study was to evaluate two different dPCR platforms for quantification of PepMV RNA: QX100 or QX200 Droplet Digital PCR from Bio-Rad (henceforth as ‘QX100/200’); and Naica Crystal dPCR from Stilla Technologies (henceforth as ‘Naica’). We took the well-characterised assays for detection and genotyping of PepMV by qPCR, developed dPCR assays, and evaluated their performances. Despite some differences observed between these two dPCR platforms, which are indicated and discussed here, the newly developed reverse-transcription dPCR (RT-dPCR) assays could be potential candidate for higher order methods for absolute quantification of PepMV.In this study, the following assays were transferred from RT-qPCR to RT-dPCR with the aim of absolute quantification without the need for standard curves: (i) for detection of all PepMV genotypes ; (ii) for detection of Peruvian and European PepMV genotypes (henceforth as ‘PepMV-Eur’); (iii) for detection of Ch2 and US2 PepMV genotypes (henceforth as ‘PepMV-Ch2’); and (iv) for detection of US1 PepMV genotype (henceforth as ‘PepMV-US1’). After the RT-dPCR assays were optimised for each dPCR platform used , their performances were validated and compared to RT-qPCR.The primers and probes for the RT-dPCR assays were the same as those used for the RT-qPCR assays. As the primer and probe concentrations and the annealing temperatures are known to influence the amplitude and clustering of the droplets, two concentrations of primers and probes were tested for the specific PepMV-Ch2, PepMV-Eur and PepMV-US1 assays, with three concentrations of primers and probe tested for the PepMV-universal assay. Additionally, different annealing–elongation temperatures were compared (from 55 °C to 65 °C). The droplet readouts demonstrated that the optimal resolution between positive and negative clusters, including the minimal amount of rain, was obtained with the same concentrations of primers and probe as used for RT-qPCR , with thThe sequences and final concentration of primers and probes used for the Naica platform were the same as for the QX100/200. However, the fluorescent labels were changed for two of the assays: from 6-carboxyfluorescein (FAM) to hexachloro-fluorescein (HEX) for PepMV-Ch2; and from FAM to cyanine-5 (Cy5) for PepMV-US1. As the Naica platform has three detection channels, the change in fluorescent labels allowed the implementation of a triplex. At the time of this experimental work, Naica provided a low throughput of 12 reactions per run, compared to QX100/200 with 96 reactions per run. Multiplexing thus increased the throughput and improved the time efficiency.https://www-s.nist.gov/dnaAnalysis/primerToolsPage.do; [A triplex assay that targeted the three different genotypes, of PepMV-Ch2, PepMV-Eur and PepMV-US1 was constructed, and its performance was compared to simplex assays. The RNA copy numbers of PepMV-Ch2 and PepMV-US1 quantified by triplex assay were significantly lower  than those quantified by simplex assays. To explain this difference, assays were analysed for primer dimer formulation using the tool available from the National Institute of Standards and Technology . PossibThe specificities of the RT-dPCR assays were confirmed through testing the RNA isolated from leaves of plants infected with different PepMV genotypes . All of these PepMV genotypes were detected with the PepMV-universal RT-dPCR . The genThe repeatability of the PepMV-genotype-specific RT-dPCR assays were evaluated through analysis of three replicates of RNA samples of different PepMV genotypes at various PepMV RNA concentrations. CVs <9% were obtained for the samples with >50 RNA copies/µL . As expeAssay reproducibility was assessed for six positive RNA samples tested in eight technical repeats on three separate days. The QX100/200 tests were also performed with different analysts and with different PCR cyclers. The mean copy numbers obtained here for the RNA samples of between 1616 and 16,896 PepMV RNA copies/µL for the different PepMV genotypes showed CVs <9%, while those with mean copy numbers between 14 and 152 PepMV RNA copies/µL showed CVs up to 22% .The sensitivities for the PepMV-universal and PepMV-genotype-specific RT-dPCR and the RT-qPCR assays were compared. The sensitivities for the RT-dPCR were a little higher for both the QX100/200 and the Naica platforms, compared to the RT-qPCR. Here, the RT-dPCR produced some signals  also at some dilutions that did not produce any signals for RT-qPCR . Similarr2) obtained by linear regression analysis showed a linear response (r2 > 0.99) of measured PepMV RNA copy numbers for all assays. Apart from the limitations presented by the assays themselves, the linear range of the RT-dPCR was limited by the number of partitions analysed. For the QX100/200 platform, the reaction mixture is divided into up to 20,000 droplets , while the Naica system generated up to 35,000 droplets . This means that the Naica system allowed quantification of higher copy number concentrations than the QX100/200 system. However, for the highest concentrations of PepMV-US1 tested , the numbers of PepMV copies were in the upper range of both instruments for quantification, at near saturation, where 100% (or ~100%) of the droplets analysed contained PepMV copies  was kindly provided by Dr J.Th.J. Verhoeven , and PepMV-Ch2 (isolate 1906) and PepMV-US1 (isolate ST 08/008) were kindly provided by Dr Inge Hanssen . PepMV isolates were propagated on Total RNA extraction was carried out for infected and healthy plant leaf material with RNeasy Plant mini kits , according to the manufacturer recommendations, with some minor modifications. Briefly, 200 mg plant material was homogenised in 900 µL lysis buffer , then 600 µL of homogenate was loaded onto RNeasy Mini Spin columns. RNA elution from these RNeasy Mini Spin columns was carried out as two consecutive applications of 50 µL  of RNase-free warm water (65 °C). Buffer controls were included with all isolations , to monitor potential contamination through the extraction procedures.The RNA of PepMV was amplified using the PepMV-universal primers and probe described by Ling et al. , and by The RT-qPCR assays were carried out with AgPath-ID One-Step RT-qPCR mix . The final reaction volume (10 µL) included 2 µL RNA sample, 900 nM primers and 200 nM probe for the specific PepMV-Ch2, PepMV-Eur and PepMV-US1 assays, or 200 nM primers and 400 nM probe for the PepMV-universal assay. Each RT-qPCR run included a non-template control and a negative isolation control. RT-qPCR was performed with 384-well plates , with reactions run as at least two technical repeats on an ABI Prism 7900HT Fast Detection system . The condition for the cycling were 10 min at 48 °C, 10 min at 95 °C, and 45 cycles of 15 s at 95 °C, with 1 min at 60 °C. Reactions were considered positive if an exponential amplification curve was produced that could be distinguished from the negative controls; with no production of an exponential amplification curve, the reaction was considered negative. Fluorescence acquisition and of Cq determination was carried out with the SDS 2.4 software . Automatic setting of the baseline was used, with manual setting of the fluorescence threshold at 0.3. This represented a level above the baseline but low enough to remain within the exponential increase region of the amplification curve.The QX100/200 RT-dPCR assays were carried out with One-Step RT-ddPCR Advanced kits for probes . The samples were each analysed as at least two technical repeats, with each run including a non-template control. The final volume of the reaction mixtures was 20 µL, which included 5 µL Supermix , 2 µL reverse transcriptase , 1 µL 300 mM dithiothreitol , forward and reverse primers, and the probe, with molecular grade RNAse-free water, and finally 4 µL RNA sample. Different final concentrations of primers and probe were compared to optimise the RT-dPCR reactions: (i) the same as for the RT-qPCR assays described above; (ii) 900 nM primers /450 nM primers  and 250 nM probe; (iii) 300 nM primers and 100 nM probe .Preparation of the reaction mixtures was carried out in 96-well polypropylene plates . These were then heat-sealed using pierceable foil, and the droplets were generated with an automated droplet generator  using automated droplet generation oil for probes  and a cartridge for automated generation of the droplets . Droplets suspensions were collected in separate 96-well polypropylene plates. After the heat sealing of these plates with droplets with pierceable foil, they were transferred to a thermal cycler . The conditions for the thermal cycling included 60 min reverse transcription at 50 °C and 10 min enzyme activation at 95 °C, which were followed by 40 cycles using a two-step thermal profile, of 30 s denaturation at 95 °C and 1 min annealing–elongation. Finally, this was followed by 10 min enzyme deactivation at 98 °C. For best possible separation of clusters of positive and negative droplets, annealing–elongation temperatures were tested between 55 °C and 65 °C. The ramp rate was set as 2.5 °C/s. Following the thermal cycling, plates were transferred into a droplet reader . A fluorescence amplitude threshold was used to discriminate the positive droplets containing the amplification products from the negative droplets, using the QuantaSoft software, version 1.7.4 . The manual setting of the threshold was to just above the negative droplet cluster, as visualised using the fluorescence amplitude versus event number in the FAM channel . SamplesThe Naica RT-dPCR assays were carried out with qScript XLT One-Step RT-qPCR ToughMix . Each sample was analysed as at least two technical repeats, with inclusion of a non-template control for each run. The final volume of the reaction mixtures was 25 µL, which included 12.5 µL ToughMix, 2.5 µL fluorescein , forward and reverse primers, and the probe, with molecular grade RNAse-free water, and 4 µL RNA sample. Simplex, duplex  and triplex (PepMV-Ch2 + PepMV-Eur + PepMV-US1) reactions were tested and compared. The final concentration of each primers and each probes used for the Naica platform in simplex, duplex and triplex reactions were the same as for the RT-qPCR assays described above.The reaction mixtures were loaded into 4-well Naica sapphire chips , and the chips were transferred into the Naica Geode  for droplet generation and PCR cycling. The droplets were generated at 40 °C, followed by thermal cycling of 10 min reverse transcription at 50 °C, 1 min enzyme activation at 95 °C, then 45 cycles of a two-step thermal profile with 30 s denaturation at 95 °C, and the 15 s annealing–elongation at 56 °C. Following thermal cycling, the chips were scanned using a Crystal reader  for the following exposure times: (i) 70 ms for FAM (blue channel); (ii) 125 ms for HEX (green channel); (iii) 25 ms for Cy5 (red channel). Data analysis used the Cristal Miner software, version 1.4.3 . After application of spillover compensation to the raw fluorescence data , a thresThe optimised RT-dPCR assays underwent validation based on the recommendations of the European and Mediterranean Plant Protection Organisation . The RT-−3 to 10−8. RNA was extracted from test plants infected with PepMV-Ch2, PepMV-Eur and PepMV-US1. The results were compared to the RT-qPCR results. For PepMV-universal assays, only the sensitivity was tested, using a dilution series  of a mix of PepMV-Ch2, PepMV-Eur and PepMV-US1 RNA-positive samples (the mix contained the same volume of RNA of each PepMV genotype). Additionally, a dilution series  of PepMV-Ch2 in RNA isolated from leaf material of healthy tomato plants was prepared and tested. Each dilution was analysed as three technical repeats for each assay. The same RNA dilutions were used for each assay.The sensitivities and linear ranges of the PepMV-genotype-specific RT-dPCR assays were tested on a dilution series (10-fold) of PepMV-positive RNA samples (in RNAse-free water), over the RNA dilution range of 10Relative quantification was carried out using the standard curve method . For comThe aim of the present study was for the development of an accurate method to provide absolute quantification of different PepMV genotypes without the need for standard curves. This was achieved by the transfer of PepMV-genotype-specific RT-qPCR assays to two dPCR platforms. In comparison with RT-qPCR, the PepMV-genotype-specific RT-dPCR assays showed sensitivities that were similar or were up to 10-fold greater, with better overall repeatability, for both of the tested platforms. With the aim of designing an assay that can detect all PepMV genotypes by dPCR, the PepMV-universal RT-qPCR assay also underwent transfer to the two RT-dPCR platforms. The PepMV-universal assay showed similar performance in dPCR and qPCR; however, the validation of the PepMV-universal assay was only carried out for the purpose of PepMV detection, and not for quantification.The high accuracy of this newly developed PepMV-genotype-specific RT-dPCR will provide for greater accuracy for PepMV RNA quantification in studies that are needed to support disease control, and for more precise quantification of PepMV in vaccines used for cross-protection. Moreover, the RT-dPCR assays can also be used for characterisation of the calibrators needed for standard curves in RT-qPCR. However, to more accurately determine the copy numbers, further studies are necessary to determine a more accurate droplet volume for the One-Step RT-ddPCR Advanced kits for probes used in combination with the QX100/200 platform, and to determine a more accurate droplet volume for the Naica system. The general unavailability of standardised reference materials represents a problem for detection and quantification of plant pathogens, and so the present study represents the first step in the development of calibrators or higher order quantitative methods for application to not only PepMV, but also to further plant viruses and further plant pathogens.


BackgroundOn April 16, 2020, the Japanese government declared a state of emergency due to the spread of COVID-19 infection, leading prefectural governors to announce a stay-at-home order for 39 days until May 25, 2020. As physical inactivity is a risk factor for osteoporosis, we investigated the short-term impact of the stay-at-home order on bone health among patients with osteoporosis in our hospital in Kanagawa prefecture.MethodsThirty patients with osteoporosis with no delays in their regular medication who received care at our hospital’s osteoporosis outpatient clinic within one month after the end of the state of emergency were included. Lumbar spine and femur proximal bone mineral density (BMD) were measured at the last follow-up date  and six (6M) and 12 months (12M) before the last follow-up using dual-energy X-ray absorptiometry. Bone alkaline phosphatase (BAP), Tartrate-resistant Acid Phosphatase 5b (TRACP5b), calcium and phosphorus were assessed at the same time points.ResultsSerum BAP concentrations were significantly lower at 0M than 12M (p=0.040), but were comparable between 0M and 6M (p=0.527). Serum TRACP5b was significantly lower at 6M than 12M (p=0.009), but was similar between 0M and 6M (p=1.000). Serum calcium and phosphorus did not differ among the time points . Similarly, lumbar spine and femoral neck BMD were comparable .ConclusionBone health in patients with osteoporosis was maintained during the short-term COVID-19 stay-at-home order among patients who experienced no delays in medication. However, larger and long-term studies are needed. In December 2019, a series of pneumonia cases of unknown cause emerged in Wuhan, Hubei, China, with clinical presentation resembling viral pneumonia [Osteoporosis is a major health problem worldwide. The disease is characterized by low bone mass and micro-architectural deterioration of bone tissue, leading to enhanced bone fragility and a consequent increase in fracture risk . PhysicaWe investigated the short-term impact of staying home on bone health among patients with osteoporosis during the COVID-19 state of emergency in our hospital in Kanagawa prefecture.EthicsThis study received ethical approval from the Ethics Review Board of Kitasato University . Informed consent was obtained in the form of opt-out on the website.PatientsThirty patients with osteoporosis  aged 71.9 ± 14.0 years (mean ± standard deviation) who received care at Kitasato University Hospital’s osteoporosis outpatient clinic within one month after the end of the state of emergency declaration were included. Of the 30 patients, 13 patients (43.3%) had prevalent fractures. All patients had been receiving osteoporosis medication without delay for at least one year in our hospital  was measured in the lumbar spine (L2-L4) and femoral neck using dual-energy X-ray absorptiometry  at the last follow-up date  and six (6M) and 12 months (12M) before the last follow-up , version 25.0. Analysis of variance with repeated measures and a Bonferroni post hoc test were used to compare the three groups. Statistical significance was defined as p<0.05.Changes in BMD and serum bone metabolism markersBMD in the lumbar spine and femoral neck did not differ among the three time points (p=0.679 and p=0.076, respectively; Figure Several scientific articles have raised an alarm on the potential detrimental effects of the COVID-19 public health emergency on patients with osteoporosis, as a reduction in bone density scanning and decreased treatment can elevate fracture risk. To examine the association of physical activity with fracture risk -9, we evPrevious studies have reported that bisphosphonates prevent bone loss during bed rest and space flights ,14. CombPatients with osteoporosis are likely to be at high risk of sequelae after contracting COVID-19. Several studies have recommended a temporary transfer to oral bisphosphonate for patients with osteoporosis because of the need for face-to-face clinical intervention for intravenous administration or subcutaneous injection of osteoporosis drugs ,17. We cWhile we observed that staying home for a short period of time had little impact on bone health, prolonged or repeated stay-at-home orders due to COVID-19 spread may affect bone health, even among patients who continue to receive their regular medications. Patients with osteoporosis are advised to engage in regular weight-bearing exercise to improve their strength, balance, posture and reduce the risk of falls ,19. HomeThere were two main limitations in the present study. First, we did not examine the activity of participants during declaration. Second, there is no control group remaining active.In conclusion, we evaluated the short-term impact of a stay-at-home order on bone health among patients with osteoporosis during a state of emergency due to COVID-19 in Kanagawa, Japan. Our results indicate that bone health was maintained during the short-term stay-at-home order among patients who experienced no delays in their regular medication. However, further investigations with larger sample size and long-term observations are needed.


To compare CT pulmonary angiographies (CTPAs) as well as phantom scans obtained at 100 kVp with a conventional CT (C-CT) to virtual monochromatic images (VMI) obtained with a spectral detector CT (SD-CT) at equivalent dose levels as well as to compare the radiation exposure of both systems.In total, 2110 patients with suspected pulmonary embolism (PE) were examined with both systems. For each system (C-CT and SD-CT), imaging data of 30 patients with the same mean CT dose index (4.85 mGy) was used for the reader study. C-CT was performed with 100 kVp and SD-CT was performed with 120 kVp; for SD-CT, virtual monochromatic images (VMI) with 40, 60 and 70 keV were calculated. All datasets were evaluated by three blinded radiologists regarding image quality, diagnostic confidence and diagnostic performance . Contrast-to-noise ratio (CNR) for different iodine concentrations was evaluated in a phantom study.CNR was significantly higher with VMI at 40 keV compared to all other datasets. Subjective image quality as well as sensitivity and specificity showed the highest values with VMI at 60 keV and 70 keV. Hereby, a significant difference to 100 kVp (C-CT) was found for image quality. The highest sensitivity was found using VMI at 60 keV with a sensitivity of more than 97 % for all localizations of PE. For diagnostic confidence and subjective contrast, highest values were found with VMI at 40 keV.Higher levels of diagnostic performance and image quality were achieved for CPTAs with SD-CT compared to C-CT given similar dose levels. In the clinical setting SD-CT may be the modality of choice as additional spectral information can be obtained. Most CT examinations are performed with intravenously applied contrast agent to increase the contrast of vessels and organs and thus the detectability of pathologies . All rouIodine = 53) as well as due to the proximity to the k-edge of iodine at 33 kVp [For all clinically available DE-CT systems, multiple usages for spectral data exist, e.g. the detection and differentiation of tumors such as head and neck tumors, renal lesion, pulmonary lesions or pancreatic adenomas . One impt 33 kVp ,29. Thist 33 kVp ,31. Witht 33 kVp . Due to t 33 kVp . HoweverThe aim of the current study was to compare CTPAs and phantom scans obtained with 100 kVp to VMI  obtained with a SD-CT using 120 kVp at equivalent dose levels. Hereby, 100 kVp images and different levels of VMI were evaluated regarding objective and subjective image criteria. Furthermore, the radiation exposure of both systems should be evaluated in a large number of patients.22.1A semi-anthropomorphic thorax phantom  was used. The phantom contains a central borehole with an insert for up to eight rods. In the present study, rods contained iodine concentrations of 0.5, 0.75, 1, 2, 5, 10 and 15 mg/mL as well as one rod with water density. The phantom has axial diameters of 300 × 200 mm, a length of 200 mm and was equipped with one extension ring, resulting in a total axial diameter of 350 × 250 mm.vol of 4.85 mGy  with tube voltages of 80, 100 and 120 kVp, respectively. Tube current was adjusted to maintain the respective CTDIvol at the given tube voltage with values of 162 mAs (80 kVp), 85 mAs (100 kVp) and 54 mAs (120 kVp). Every scan was repeated three times and in-between scans, the phantom was repositioned. For 120 kVp, monoE-40, monoE-50, monoE-60 and monoE-70 images were calculated. All images were reconstructed with a slice thickness of 1.0 mm.The phantom was scanned  using a CTDII is the intensity measured in the iodine rod, SW is the intensity of the water rod and δW is the standard deviation measured in the water rod.Regions of interest (ROI) with an area of 1 cm² were analyzed in 3 different slices for each iodine concentration in each scan and for each image type. In total, 486 ROIs resulted. For each ROI, intensity value was measured in Hounsfield Units (HU). For each ROI, CNR was calculated by: 2.2Institutional review board (IRB) approval was obtained for this retrospective study including all protocols . Informed consent was waived by the IRB as no additional data besides clinical obtained images were used. All examinations were performed exclusively for diagnostic use and were performed only with clinical standard protocols. All patient data were completely anonymized at the beginning of the study.vol for all patients with suspected PE as 4.85 mGy. So, this dose level was selected as the sighted mean dose level of the reader study patients as well. Patients were selected without knowledge of the respective images to avoid any bias. Patients were examined using a conventional spiral CT-system  or a spectral-detector CT-system . For C-CT, only imaging data of examinations with a tube voltage of 100 kVp was used. For SD-CT, spectral information was obtained from spectral base image (SBI) datasets . Mean age for patients scanned with C-CT was 59.4 years (27–86) and 65.9 years (33–95) for patients scanned with SD-CT. PE was classified as central, segmental or subsegmental; PE in multiple locations was also possible  was selected retrospectively for the imaging analysis. Hereby, the data was selected to match similar average radiation doses for C-CT and SD-CT. At the timepoint of the beginning of the reader study, the man CTDIpossible .Table 1PFor the reference standard, the clinical report as well as a repeated reading of the images by the author was used. Examinations not suitable for clinical evaluation (i.e. due to contrast phase or motion artefacts) were excluded based on the clinical report. Also, examinations with other severe findings  were excluded to minimize recall bias and to focus on the image quality regarding diagnosis of PE.2.3vol was selected via automatic dose control for all patients (C-CT and SD-CT). A tube voltage of 100 kVp corresponds to a mean energy of ∼60 keV and a tube voltage of 120 kVp corresponds to a mean energy of ∼70 keV.The same scanning protocol was used for both CT-systems. Scans were performed with 60 mL of intravenous contrast agent  followed by a 50 mL saline chaser with an injection rate of 3.5 mL/s. The scan range covered the whole thorax, was performed in inspiration and was chosen by an anteroposterior scout. The bolus tracker was placed within a region-of-interest (ROI) in the pulmonary trunk (threshold for scan start: 100 HU). The scan was performed craniocaudally with a pitch of 0.9 and a 128 × 0.625-mm (C-CT) or 64 × .0.625 mm (SD-CT) detector coverage. Obese patients were examined using a tube voltage of 120 kVp for both systems. Non-obese patients were examined with 120 kVp for SD-CT compared to 100 kVp for C-CT. CTDITomographic slices were obtained with a field of view of 350−500 mm, based on the diameter of the patient. A 512 × 512 image matrix with a slice thickness of 0.9 mm was used for axial thin slices data.2.4vol) and dose length product (DLP) were collected. By multiplication of DLP by the chest conversion factor (0.0145), the effective dose (ED) could be calculated [For every patient, the automatically generated dose protocol was extracted after the examination. Tube voltage (kVp), tube current (mA), volume-weighted CT dose index  with 40 keV, 60 keV and 70 keV were calculated using the commercially available spectral workstation , Philips Healthcare, The Netherlands). Thus, the following datasets were obtained:1) 100 kVp using C-CT.2) monoE-40 (VMI with 40 keV), monoE-50 (VMI with 50 keV), monoE-60 (VMI with 60 keV) and monoE-70  using SD-CT.2.5Each dataset was independently evaluated by three blinded radiologists , the author not being a reader. To avoid a recall bias, only 30 cases were evaluated at a single timepoint with a minimum of two weeks between the sessions. Cases were presented in a randomized order, i.e. case number and type of dataset - C-CT/monoE-40/monoE-60/monoE-70 - was randomized. Image parameters  could be individually chosen by the readers. Subjective image quality was rated regarding the following criteria:1- not diagnostic; 2 - sufficient; 3 - satisfactory; 4 - good; 5 - very good; 6 - excellentContrast of pulmonary vessels was rated. Hereby, central pulmonary vessels (pulmonary trunk and main pulmonary arteries), segmental pulmonary arteries and subsegmental pulmonary arteries were analyzed. For subsegmental arteries, four different regions were rated  to avoid influence due to a partially decreases/increased contrast in singular subsegmental vessels. Contrast was rated using the following scale:1 - not diagnostic; 2 - sufficient; 3 - satisfactory; 4 - good; 5 - very good; 6 - excellentThe presence of streaking artefacts was assessed using the classes:1 - massive artefacts (image not diagnostic); 2 - major artefacts; 3 - minor artefacts; 4 - no artefacts.2.6Diagnostic confidence regarding detection of PE was evaluated for each localization  separately. Hereby, confidence was rated using the following scale:N) and ratings of 4–6 as positive for PE (PEP). PE should only be rated as positive in the specific location when a clear embolus was visible.1 - no PE, completely confident; 2 - no PE, probably confident; 3 - no PE, poor confidence, additional imaging needed; 4 - PE present, poor confidence, additional imaging needed; 5 - PE present, probably confident; 6 - PE present, completely confident. Ratings of 1–3 were considered negative for PE . Continuous data are expressed as arithmetic mean ± SD. Data were tested for Gaussian distribution via D'Agostino-Pearson omnibus test. As Gaussian distribution was present, two-sided paired 33.1CNR in the phantom study was significantly higher in monoE-40 compared to other VMI and to C-CT . CNR was3.2vol was higher for C-CT (5.91 mGy) compared to SD-CT (5.23 mGy). For all scans with 100 kVp (non-obese patients), mean CTDIvol was 4.60 mGy for C-CT whereas it was 4.35 mGy for all non-obese patients with SD-CT  and 534 (SD-CT) were obese. Mean CTDIth SD-CT . Each covol of the selected patients in the reader study was 4.85 mGy for both C-CT and SD-CT. For C-CT, a standard deviation of 1.22 mGy (range 3.3–8.1 mGy) was found, whereas for SD-CT the standard deviation was 1.74 mGy (range 3.2–9.7 mGy).The CTDI3.3A comparison of images obtained with the C-CT (100 kVp) and with the SD-CT  is shown in Subjective contrast level of pulmonary vessels was highest in monoE-40 images for all subsections; however, lowest subjective image quality was shown for those image type. Highest subjective image quality was shown for monoE-60 and monoE-70 images with significantly higher image quality compared monoE-40 and 100 kVp . No sign3.4Diagnostic confidence was high or medium in most cases when using monoE-40 images . As expeSensitivity was higher than 90 % for all locations of PE for all image types, except monoE-40 . For cenSpecificity was higher than 90 % for all image types and locations of PE, except for subsegmental PE at 100 kVp . For acc4In this work, conventional CT scans obtained at 100 kVp were compared to virtual monochromatic images calculated from spectral data obtained with a spectral detector CT at 120 kVp. Both approaches were evaluated regarding objective and subjective image criteria using phantom scans and retrospectively collected in-vivo patient data. The results demonstrated that both methods enabled excellent diagnostics for detection of PE at an equivalent dose level. Hereby, SD-CT offers additional capabilities, including iodine quantification, reduced beam-hardening artifacts and increased iodine signal via low-keV VMI. Additionally, lower radiation doses were found for SD-CT compared to C-CT when evaluating over 2000 examinations.In the current study, a sensitivity of 100 % for central PE could be reached with all VMI datasets as well as with C-CT. The best results regarding sensitivity and specificity of segmental and subsegmental PE could be found when monoE-60 and monoE-70 were used. For sensitivity, C-CT was superior to monoE-40, whereas the opposite was found for specificity. These results correspond with the results of subjective image quality where the best results were also reached with monoE-60 and monoE-70. This is most likely as in clinical routine, radiologists are used to these image types. As different patients were used for SD-CT and C-CT, the diagnostic performance of both systems cannot be compared to the full extend. A recall bias is thinkable as for monoE-40, monoE-60 and monoE-70, the same patient data were used. To minimize possible recall bias, readers were advised to evaluate a maximum of 30 cases during one reading session with a minimum of two weeks between two sessions. The order of cases was randomized, so that no VMI should be favored, even if a recognition of cases was present. Overall, the present results indicate that SD-CT is equivalent or even superior to C-CT regarding subjective image quality and diagnostic performance at an equivalent dose level. For image quality and subjective contrast, small standard deviations were found, indicating consistency in the respective groups and between the readers. Despite the small standard deviations, a low interobserver agreement was found. This is most likely due to a narrow range but not identical ratings and the use of Fleiss’ kappa, calculating the agreement of all three raters at once. However, given the small standard deviations, the results seem consistent and reliable.The results of the diagnostic performance do not correlate to the diagnostic confidence and subjective contrast reported by the readers. This could be as radiologists correlate a high contrast to a high diagnostic confidence. However, a strong increase of the iodine signal does not seem to increase the diagnostic performance - at least in the current study where a sufficient contrast of the pulmonary vessels was present in all cases. Additionally, the diagnostic performance could be lower for monoE-40 due to a lack of experience with the datasets. Another factor for the diagnostic superiority of monoE-60 and monoE-70 compared to monoE-40 could be the examined patient type. As a 100 kVp protocol was used for C-CT, only non-obese patients could be included. As the radiation dose should be matched with SD-CT, obese patients were not included for this system as well. In obese patients, where image quality is degraded at low-kVp levels, monoE-40 could be beneficial due to the boost in the iodine signal. However, as there is an increased rate of photon starvation for low-energy photons, monoE-40 images could also show decreased image quality in obese patients. A main advantage of monoE-40 images is the possibility of a boost in the iodine signal when there is reduced contrast of the pulmonary vessels.With conventional image reconstruction (i.e. filtered back projection) the image noise significantly increases with low tube voltages or low keV VMI settings. Our CNR measurements indicated that this noise related effect did not disrupt the boost in the iodine signal. Iterative reconstruction algorithms enable the dose efficient applicability of both modalities by suppressing the increased noise level ,38. ThisIn the current study, radiation exposure was significantly lower with SD-CT compared to C-CT for each comparison . This shows that despite concerns regarding radiation exposure, it may even be lower for SD-CT compared to conventional CT-systems. This partly might be due to improved protocols which were adapted for the new system. However, we constantly optimize our protocols of each system to minimize radiation exposure so that the observed reduced radiation exposure seems realistic. Certainly, SD-CT systems do not go along with an increased radiation exposure compared to C-CT, not even if a 100 kVp protocol is used for the latter.DE-CT enables the generation of iodine maps for the assessment of the lung perfusion increasing the sensitivity for the detection of PE ,40. SD-CThe presented study design is subject to limitations. Firstly, the performance and radiation dose levels are strongly depending on clinical applications. Our results are specific for PE diagnostics, and further studies are essential to evaluate other applications. Additionally, obese patients could not be evaluated as those are not examined using 100 keV. Further, different patients were used for the two CT systems. Despite matching dose levels and excluding non-diagnostic examinations, an influence of the patient selection to the results could be possible. In the current study, a combination of VMI and iodine maps was not evaluated. This combination could yield even higher sensitivity, specificity and diagnostic accuracy compared with VMI alone or compared to 100 kVp examinations. This evaluation should be subject of further studies. Despite the stated drawbacks the current study gives an essential indicator that either approach can be potentially employed with high diagnostic performance and without major drawbacks.5In conclusion, this study illustrates that detection of PE is enabled for non-obese patients at an equivalent diagnostic level and a similar radiation exposure with SD-CT compared to C-CT at 100 kVp. With SD-CT even reduced radiation doses were found for obese and non-obese patients. In the clinical setting where spectral results - such as material specific maps or VMI - are useful, SD-CT may be the modality of choice.Institutional review board (IRB) approval was obtained for this retrospective study including all protocols . Informed consent was waived by the IRB as no additional data besides clinical obtained images were used. All examinations were performed exclusively for diagnostic use and were performed only with clinical standard protocols. All patient data were completely anonymized at the beginning of the study.The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.German Department of Education and Research (BMBF) under grant IMEDO (13GW0072C), the German Research Foundation (DFG) within the Research Training Group GRK 2274 and the German Research Foundation (DFG) within the project 432290010. Funding had no influence on the results of this study.This work was supported by the Andreas P. Sauter: Conceptualization, Data curation, Formal analysis, Methodology, Supervision, Validation, Visualization, Writing - original draft. Nadav Shapira: Conceptualization, Methodology, Writing - original draft. Felix K. Kopp: Investigation, Software, Methodology, Writing - review & editing. Juliane Aichele: Investigation, Writing - review & editing. Jannis Bodden: Investigation, Writing - review & editing. Andreas Knipfer: Investigation, Writing - review & editing. Ernst J. Rummeny: Funding acquisition, Project administration, Writing - review & editing. Peter B. Noël: Funding acquisition, Methodology, Project administration, Validation, Writing - original draft.NS is an employee of Philips GmbH.The remaining authors of this manuscript declare no relationships with any companies, whose products or services may be related to the subject matter of the article.


Incineration has emerged as one of the acceptable ways to treat municipal solid waste (MSW) due to its potential in reducing the mass and volume of the waste. However, it produces two major by-product residues, namely MSW-bottom ash (MSW-BA) and MSW-fly ash (MSW-FA). These residues have gained great attention to their hazardous nature and potential to be reused and recycled. In this paper, the physicochemical characterizations of the MSW-BA and the MSW-FA were performed, followed by a systematic investigation of metals extraction from MSW-BA and MSW-FA. Various extracting agents were used to investigate the possibility to extract 21 metals including cadmium (Cd), vanadium (V), chromium (Cr), and lead (Pb). It was revealed that some metals were present in a high amount in the MSW-BA while other metals were higher in the MSW-FA. Moreover, the energy-dispersive X-ray spectroscopy results revealed that the MSW-BA was dominated by oxygen (O) 55.4 ±0.6 wt%, silicon (Si) 22.5 ±0.3 wt%, and calcium (Ca) 18.5 ±0.2 wt%. On the other hand, the MSW-FA was enriched with Ca 45.2 ±0.5 wt%, and O 40.3 ±0.4 wt%. From the scanning electron microscopy, the MSW-BA was observed as flaky with an irregular surface that consisted of large pores, while, the MSW-FA was present as agglomerated particles and had a bimodal distribution. Moreover, Fourier transform infrared spectroscopy revealed that Al-Fe-OH, Al-Al-OH, Si-O, C-O, and C-H were some of the major functional groups present in the ashes. The F-tests concluded that the metal extraction from the MSW-BA and MSW-FA were significantly affected by the acid type. it is concluded that nitric acid and phosphoric acid were the best-suited acid for the MSW-BA while sulfuric acid and phosphoric acid for the MSW-FA. More than 11 wt% of Cd and 9 wt% of Cu were extracted from MSW-BA while 6 wt% of Pb and 4.5 wt% of V were extracted from the MSW-FA. The present methodology is an interesting development in metal extraction from the MSW-BA and the MSW-FA, which can develop in a cost-effective and sustainable option to utilize MSW. The management of manuscript solid waste (MSW) has become one of the major issues around the world and is, therefore, a key concern for municipal societies. A successful MSW management should execute proper management control, including planning, assortment, transportation, treatment, extraction, recovery recycling, and disposal. One of the most predominant practices used to treat MSW is incineration, which uses innovative treatment solutions to produce green energy and reduce the volume and mass of MSW up to 80% and 90% respectively ,2,3. HowWaste re-utilization is a promising way towards developing an environmentally friendly and cost-effective products –14. InciMetal extraction using different extraction agents is one of the best techniques used to investigate the metal-binding mechanisms within solid residues . It is wFor a small country like Qatar, the generation of the MSW is as high as 4,500 tons per day ,23–25 anIn this study, the physicochemical characterizations of the MSW-BA and MSW-FA from one of Qatar’s MSW incineration plants were studied. Also, the best suited extracting agents were determined by using several agents, namely (i) inorganic salt extracting agents, including sodium acetate, diammonium ethylenediaminetetraacetate (EDTA) , ammonium oxalate, ammonium sulfate, ammonium nitrate, (ii) acidic extracting agents such as nitric acid, phosphoric acid, perchloric acid, and sulfuric acid, and (iii) alkaline extracting agents such as sodium carbonate and sodium hydroxide to extract metals from MSW-BA and MSW-FA.Because of the complexity of the MSW-BA and MSW-FA, replicate samples were collected at different months for one year. The MSW-BA and MSW-FA were freshly collected in representative 5 kg samples from a local incinerator (Qatar Company: Domestic Solid Waste Management Centre (DSWMC)–Doha, (State of Qatar). The permission of the sample collection was already granted by the Company. We confirm that the field studies did not involve endangered protected species. The sample was dried at 100°C for 24 h, ground at different particle sizes , and sieved through a standard sieve to obtain homogenized ashes. Then, the sample was kept in a clean and isolated glass bottle. The particle size used in this study was 0.125–0.250 μm to carry out all the analytical work.® using a sequence of mineral acids  [solution) was determined by preparing aqueous extracts at 1:1 ratios of solid: distilled water (w/v) and mixing it at room temperature for 24 h using a stirrer. The samples were then filtered using a 0.45 μm pore membrane and the filtrate pH was recorded using a pH meter (HQ440d multimeter).Total acid digestion was performed to determine the total metal content present in the MSW ashes according to USEPA SW846 Method 3050B . 1 g of dried powdered ash was carefully digested at 90°C in Teflonic acid)  followedIon chromatography (IC)  was used to determine the ion content presents in the MSW-BA and the MSW-FA.The determination of the mineralogical composition of the MSW-BA and the MSW-FA was identified by powder X-ray diffraction (XRD) . The scan was run from 5 to 85 .−1. The pellets were prepared by mixing 1 mg of powdered samples with 300 mg of potassium bromide.FTIR was used to identify the functional group on the surface of the MSW-BA and the MSW-FA using the PerkinElmer 400 Spectrum instrument using UATR  and spectra ranged from 400 to 4000 cmScanning electron microscopy- energy-dispersive X-ray spectroscope (SEM-EDX)  was used to determine the morphology of the MSW-BA and the MSW-FA.50, which is an important aspect as it tells where the 50% of the cumulative particle size lies. While Dw gives a better understanding of the particle size on the volume occupied, and particle distribution (PD) explains how particle size is similar to the particle distribution. Dw and PD were calculated by using  was used to determine the particle size distribution for each fraction. The median of each particle size is denoted by Dsing Eqs –3.Ni is the number of particles at the i th class in the size-distribution histogram.Where For the Brunauer-Emmett-Teller (BET) surface area, pore radius and pore volume, the surface area analyzer  was used.The metal extraction from the MSW-BA and the MSW-FA was carried out using three types of extraction media: (i) inorganic salt, (ii) acidic extracting agents, and (iii) basic extracting agents. Two influencing factors were taken into account, including the extracting agent concentration  and the solution temperature (namely 25°C and 60°C). 1.0 M stock solution was prepared for all the extracting agents and then diluted to 0.1 M and 0.5 M, The “liquid to solid” “L/S” ratio was kept constant at 50 mL/0.05 g. Then the optimal combination was determined by the range analysis. Scheme 1  shows th0.05 g of the MSW-BA and 50 mL of the prepared extracting agents as mentioned in Scheme 1  were addppt). The solubility product constant, Ksp, is the equilibrium constant used to describe the dissolution of a solid in a solution. The lower the Ksp of a solvation reaction, the lesser the thermodynamic favorability of dissolving that solid in a solution. More details are discussed in section 2.2.4. The study did not consider the effect of temperature on Ksp. The value of Ksp at 25°C was only considered.The pH of the extracting agent is a key parameter that strongly affects the extraction efficiency of metal from the ash. The possible extraction mechanisms that may occur at neutral pH involves metal hydroxide precipitation. Eqs For dictations:For trications:m and Ksp are the metal concentration (in molarity (M)) and solubility product constant, respectively.Where CMicrosoft Excel was used to perform a two-way Analysis of variance (ANOVA) double factor. The precision of the instrument obtained from ICP-OES was obtained by performing the F-test.All costs are expressed in US dollars (USD) considering the USD quotation of December 2019. When 1 USD was equivalent to 3.64 QR.  . To asse4 ± 2178), Fe (3.65×104±548), Al (2.93×104 ± 439), Mg (1.09×104±163), K (6.69×103±100), and Pb (52 ±1) were the major content present in MSW-BA whereas Cd (5.98 ±0.09), Ba (567± 9), Li (17.5±0.3), V (2.81 ×103±42), and Sb (498 ±7) were present in the least amount. On the other hand, the MSW-FA was mostly dominated by Al (4.98×104 ± 747) Ca (2.04×105 ±3053), Na (1.18×104 ± 620), K (1.14×104± 171), Mg (1.60×104 ± 240), and Fe (1.33×104±200), while As (6.58 ± 0.10), Ba (696 ± 10), Mo (8.52 ± 0.18), Ni (41.5 ± 1.2), and Li (11.9 ± 0.2) were present in the least quantities. The findings were similar to Kowalski et al. [i et al.  and Parki et al.  The pres5±193 mg/L, 129.0 ±0.1 mg/L, and 19.1±0.0 mg/L, respectively). The IC confirmed that the MSW-FA had a very high concentration of chloride, almost double in contrast with the MSW-BA. High Cl and Na presence can be associated with high salt food intake, while potassium presence can be associated with high fertilizer consumption [Ion chromatography analysis (IC) confirmed the presence of chloride more than 160.0 ± 0.2 mg/L followed by sodium and potassium . Moreover, the IC obtained for the MSW-FA also showed a high dominance of chloride, sodium, potassium, and sulfate  technique was used to study the surface area and pore size distribution of the MSW-BA and the MSW-FA. The surface area of the MSW-BA was 49.29±0.05 mparticle . This man et al.  found MSg et al.  investigg et al. , found t-1, 875 cm-1, and 936 cm-1 indicated the presence of Al-Fe-OH. At 1103 cm-1, another vibration indicated the presence of the Si-O bond and Si-O-Si stretching vibration [-1, indicating the presence of carbonates of O-C-O [-1, the peak signifies the presence of C = O, while a broad peak at 3400 cm-1 was observed, which possibly indicates hydroxyl (O-H) stretch [-1 which indicates the presence of silica Si-O. The peak at 1410 cm-1 indicated the presence of C-O. The incineration process might have not been completed as the peaks at 2940 and 2842 cm-1 indicate the presence of CH2 asymmetric and CH3 symmetric stretching vibrations, respectively [-1 and 2358 cm-1, which represented the stretching, and deformation of OH and H-O-H groups, respectively [-1 and 1450 cm-1 suggesting a greater contribution in water-bound and carbonates. However, the main absorption band was noted at 940 cm-1 that was assigned to asymmetric stretching vibration for Si-O.ibration ,53. A stof O-C-O . Lastly, stretch . On the ectively . A broadectively . AdditioStudies have suggested that the leaching behavior of metals is dependent on various parameters including bulk properties of hosting particles, their mode of occurrence, and the pH of the leaching solution . The extWhere HA denotes the protonated extracting agent.When comparing coarse ash to fine particles, fine particles that have larger surface areas are exposed to leaching solution and release a greater amount of metals due to surface contact, while lower surface area promotes adsorption of metal. Nevertheless, a decrease in the particle size promotes the adsorption of metals. Major elements found in the ash include Si, Al, Ca, and Fe that affect the ash properties as well as mineralogy, which play a crucial part in the leaching of toxic metals ,58.The metal’s properties, namely electronegativity, solubility, and atomic radii affect the metals extraction and the maximum acid reachable during the ash extraction processes in addition to the metal removal kinetics. The extraction efficiency of metals from the ash could be due to different mechanisms: (i) solubilization by acid desorption, (ii) extraction by bond breaking from the ash matrix, and (iii) water/acid stripping. The electronegativity, solubility, and atomic radii of the metals are correlated with the activation energy required for the metals desorption from the ash matrix due to the affinity of the metal to the ash particle. In the extraction process, the first step would be rapid metal removal due to the effect of water/acid stripping, while, the second one is the internal diffusion phenomena. Also, the availability of the metals bound residues in the ash particle, which persist in the ash matrix, may considerably reduce the accessibility and the availability of the acid extraction process. These bound residues in the ash matrix could be described as covalently bound to the ash particle, adsorbed residues to the ash matrix by reversible non-covalent interactions; and entrapped residues, which are retained within the ash matrix. The ashes by nature are basic, thus when basic solutions were used to extract metals, the extraction of the metals was almost undetectable. This could be explained due to the precipitation and/or sorption of the solution at high temperatures, making the extraction process unsuccessful. Therefore, it was concluded that the basic solutions were not feasible for the optimum extraction of metals.2- is a conjugate base, however, it requires basic conditions to dissolve the carboxyl group present in EDTA which makes the solution of diammonium-EDTA basic [Inorganic salt also exhibited similar results as basic extracting agents. For instance, sodium acetate, which is an inorganic salt, but when dissolved in distilled water becomes basic with a pH greater than 7. Similarly, EDTATA basic . Ammoniu2O, SiO2, AlO3, Fe2O3, and CaO. Disassociation of such minerals in a reducing environment will most likely lead to the release of the desired metals. The results are expressed in terms of the percentage of metal extraction using various acids i.e. perchloric acid, phosphoric acid, sulfuric acid, and nitric acid. Various concentrations of the previously-mentioned acids were tested as well as the effect of temperature. Under the acidic environment, the metals were drastically leached in contrast with the alkaline solution. This can perhaps be explained due to the neutralization process followed by the disassociation of oxides and carbonates which were formed earlier during the incineration process as discussed earlier. A redox reaction might also have played a vital role in releasing some of the metals in the aqueous solution. As observed from XRD various meals were present in their mineral forms such as K3/H2O2/HF acids. The water-soluble dilatation was introduced which was expected to collect some metals including Ca, Pb, Na, and K but it was found 20 6 of MgCl2 was required to separate the metals.On the other hand, the MSW-FA showed a similar trend; however, it was observed in almost all the metals, a high temperature yielded the highest percentage removal. Cu, Cr, Fe, Zn, and Pb gave the highest percentage using phosphoric acid  at 60°C. A 4.6 wt% of V was extracted using phosphoric acid at 0.5 M and 60˚C. It is important to highlight one of the reasons why perhaps the MSW-FA had the lower percentage removal in contrast with the MSW-BA; it could be due to the initial high concentration of metals present in the raw ashes. It was observed that in some cases, it was easier to extract some metals from the MSW-BA while in other cases; it was easier to extract from the MSW-FA. This perhaps can also be associated with the chemical bonding between ashes and the metal. Some metal binding was so strong, it was impossible to disassociate the metals while in some cases the bonds were weak. Al-Ghouti et al.  partiallThe extraction of metals from the ash is very complicated as different metals reacting differently with the extraction agent, which gives different efficiency rate. Studies suggest various techniques that were found successful in other regions. Though there are various other methods applied in other studies that utilize various complex techniques to extract metals, however, this paper explored different options; aiming to outline a method to successfully extract maximum metals from the ashes. The results were compared with few studies, which explored the possibility to extract metals ,56.Analysis of variance (ANOVA) was used to validate the data and understand the effect of concentration on the metal extraction. If the F-value is greater or equal to F-critical and P-value is less than or equal to 0.05, then the data is significantly different amongst the mean of groups  . The ressp values adapted from Lide (2006) [sp, it can be assumed that in general for both MSW-BA and MSW-FA, PbCO3 will precipitate before BaCO3 as Ksp for the former is lower than the latter . On the other hand, in the MSW-FA, Al would also precipitate first at pH 3.56 followed by Cr, Cu, and Zn. From ppt of metals varies from one metal to another. Based on the data given in 2(CO3)3 is soluble in solution and; therefore has no Ksp likewise Cr2(CO3)3.e (2006)  and precThe cost of metal extraction from ash is a key component in promoting and application of the proposed extraction procedure. It is a common method used in decision making and predicting the possible environmental effects in the overall application process. The cost needs to cover operating expenses, including chemical and consumable cost, multiple iterative assessments, batch process optimization, and the number of experiments, electricity cost, and others. The complete process of the metal extraction was carried out in a lab-scale. Hence, the cost analysis was performed considering lab-scale metal extraction.To propose the cost analysis, a detailed analysis was established by including the following factors, the cost of the acid material, energy consumption, miscellaneous expenses, and other laboratory efforts to treat a given amount of ash. This model allowed for different sizes of the batch process to be analyzed for overall efficiency and cost that could treat 1 kg of ash input.For the metal extraction, it was concluded that nitric acid and phosphoric acid were the best-suited acids for the MSW-BA while sulfuric acid and phosphoric acid for the MSW-FA. Based on this conclusion, the breakdown of the cost of each step of the metal extraction process for the MSW-BA and the MSW-FA is presented in 2O SiO2 AlO3 Fe2O3, CaO were amongst the major crystals present in both ashes. Particle size distribution revealed that the MSW-BA was heterogeneous with a variety of particle size distributions, while the MSW-FA was found to be more towards bimodal distribution. Al-Fe-OH, Al-Al-OH, Si-O, C-O, and C-H were amongst some of the major functional groups found in both ashes.The present study aimed to investigate the physicochemical characteristics and potential of metal extraction from MSW-BA and MSW-FA that are generated from one of the incineration plants in Qatar. Morphologically, the MSW-BA was heterogeneous, flaky, and powdery indicating low strength. However, the images also revealed that the MSW-BA was rough, irregular, and angularly shaped yet closely packed together. On the other hand, the MSW-FA was observed to be looser and finely distributed as evident from their structure, the ashes were more rode shaped and elongated. Both ashes were found rich with various metals including Fe, Al, Mg, and Pb in MSW-BA while Al, Ca, Na, K, Mg, and Fe in MSW-FA. Furthermore, KIt was found that both ashes preferred acidic solutions as a high leading to a high percentage of metals being extracted from the MSW-BA and the MSW-FA. The study also explored the possibility to extract various metals from the MSW-BA and the MSW-FA using various extraction agents. This study found the acid solutions were effective for metal extraction. More than 11 wt% of Cd and 9 wt% of Cu were extracted from the MSW-BA, while 6 wt% of Pb 4.5 wt% of V was extracted from the MSW-FA with only 6 h contact time. The overall extraction yield of metals in aqueous phases can be used for further metal extraction using a leaching-extraction procedure with a longer duration.Furthermore, the metal extraction from the MSW can be an effective way to recycle the ashes. The present technique is an interesting development in metal extraction from MSW-BA and MSW-FA, which can develop in a cost-effective and sustainable option to utilize Qatar MSW. However, further studies are required for the upscaling of the solvent leaching technique to obtain maximum metal extraction with minimum energy utilization. This work can contribute to future work involving the utilization and application of problematic MSW-BA and MSW-FA.S1 Graphical abstract(TIF)Click here for additional data file.


Accumulating evidence shows that circRNAs play critical roles in the development of human tumors. We observed that circ_0000527 was overexpressed in osteosarcoma cells  compared in hFOB1.19 cells. We demonstrated that the circ_0000527 level was higher in osteosarcoma specimens than in non-tumor specimens. The ectopic expression of circ_0000527 was shown to induce cell growth, cell cycle progression and the secretion of inflammatory mediators, including IL-1β, IL-6, IL-8 and TNF-α. We demonstrated that circ_0000527 sponges miR-646 in osteosarcoma cells and that ARL2 is a target gene of miR-646. MiR-646 expression was decreased and ARL2 was overexpressed in osteosarcoma cells  compared to hFOB1.19 cells. Overexpression of circ_0000527 was demonstrated to induce ARL2 expression in MG-63 cells. We showed that miR-646 was downregulated in osteosarcoma specimens compared to that of non-tumor specimens and that the level of circ_0000527 was negatively correlated with miR-646 expression in osteosarcoma specimens. The elevated expression of circ_0000527 was shown to promote cell growth and cell cycle progression by modulating miR-646 expression. The ectopic expression of circ_0000527 was shown to promote cell growth, cell cycle progression and the secretion of inflammatory mediators by modulating ARL2. The present study suggested that the circ_0000527/miR-646/ARL2 axis may be a potential treatment target for osteosarcoma. Osteosarcoma is the most frequent type of primary malignant bone tumor in adolescents and children –5. The pAccumulating evidence has shown that circRNAs can modulate gene expression by acting as competing endogenous RNAs (ceRNAs) for miRNAs and their target genes –21. CircWe demonstrated that the circ_0000527 level is higher in osteosarcoma specimens than in non-tumor specimens. We showed that the ectopic expression of circ_0000527 induces cell growth, cell cycle progression, invasion and secretion of inflammatory mediators, including IL-1β, IL-6, IL-8 and TNF-α.We observed that circ_0000527 was overexpressed in osteosarcoma cells  compared to hFOB1.19 cells . MiR-646Then, we determined the expression of circ_0000527 in the osteosarcoma specimens. We observed that the circ_0000527 level was higher in the osteosarcoma specimens than in the non-tumor specimens .The level of circ_0000527 was upregulated in 20 cases  compared to the non-tumor specimens .The level of circ_0000527 was upregulated in MG-63 cells after treatment with pcDNA-circ_0000527 compared to that of the control group . The eleThe elevated expression of circ_0000527 induced IL-1β expression in MG-63 cells . The oveAs predicted by Starbase, circ_0000527 might be regulated by miR-646 . The levAs predicted by Targetscan, ARL2 expression might be regulated by miR-646 . We obseFurthermore, we demonstrated that miR-646 was downregulated in osteosarcoma specimens compared to non-tumor specimens . The levThe elevated expression of miR-646 decreased expression of CDK2  and ki-6The level of ARL2 was downregulated in MG-63 cells after treatment with ARL2 siRNA compared to that of the control group . KnockdoKnockdown of ARL2 suppressed IL-1β expression  and IL-6in vitro and to decrease cancer growth in vivo partially by regulating miR-936/HMGB1. Lin et al. [Increasing evidence has shown that circRNAs play critical roles in the development of human tumors –33. Exisn et al.  demonstrn et al. . Wu et an et al.  indicaten et al.  illustran et al.  illustran et al.  also obsn et al. . We obsePreviously published studies have shown that circRNAs can act as ceRNAs of miRNAs to decrease the activity of miRNAs and modulate gene expression, transcription and binding with these proteins , 46. ForIn summary, this study revealed that circ_0000527 is overexpressed in osteosarcoma cells and specimens and that ectopic expression of circ_0000527 promotes cell growth, cell cycle progression, invasion and the secretion of inflammatory mediators by modulating ARL2. Therefore, the circ_0000527/miR-646/ARL2 axis may be a potential treatment target for osteosarcoma.Osteosarcoma cells  and hFOB1.19 cells were acquired from the American type culture collection (ATCC) and cultured in DMEM  supplemented with streptomycin, penicillin and FBS. miR-646, pcDNA circ_0000527 and their controls were synthesized at Shanghai GenePharma. Cell transfection was performed with Lipofectamine 3000 . Osteosarcoma surgical specimens and control specimens were obtained from Nanyang First People’s Hospital following the criteria approved by the institutional review board. Informed consent was gained from each patient.-DDCt was utilized for analysis of the PCR results.Total RNA from cells cultured from the osteosarcoma specimens was collected using a TRIzol kit . The relative expression of circ_0000527, miR-646 and ARL2 was detected by a qRT-PCR assay using a SYBR Green kit on a Bio-Rad CFX96 PCR system (VisonBio Scientific). U6 snRNA and GAPDH were used as the controls for the miRNA and mRNA, respectively. The method of 2The cells were cultured in 96-well dishes for 0, 24, 48 and 72 hours. At the desired time point, 10 ul of CCK-8 solution from the kit was added into each well. After 4 hours, the OD at 450 nM was assessed. Cytokines in the supernatant were assessed by ELISA. IL-1β, IL-6, IL-8 and TNF-α kits  were used to determine the IL-1β, IL-6, IL-8 and TNF-α concentrations, respectively. For the cell cycle analysis, the cells were stained with propidium iodide (PI) for half an hour. The cell cycle was assessed by flow cytometry.The Magna RIP RBP Immunoprecipitation Kit (Millipore) was used for the RIP assay. The cells were resuspended in the lysis buffer containing RNase and protease inhibitors (Epicentre). The cell lysate was then incubated with beads coated with an AGO2 antibody or a control IgG. After incubation in proteinase K, the immunoprecipitated RNA was isolated and assessed by qRT-PCR.The wt and mut constructs of circ_0000527 and the ARL2 gene were amplified with RT-PCR. The cells were cultured in 24-well dishes and co-transfected with the miR-646 mimic or miR-NC and pGL3-circ_0000527, ARL2-3’UTR-WT, pGL3-circ_0000527 or ARL2-3’UTR-MUT and pRL-TK by Lipofectamine 3000 . After 48 hours, the firefly and Renilla luciferase values were detected using a dual-luciferase kit . Renilla luciferase was used as the control.All results are shown as the mean ± standard deviation. The significant difference between two groups was determined with a Student’s t test. P <0.05 was set as statistically significant.


Assess the stability of three different mini-implants, based on thread shapefactor (TSF), and evaluate stresses at the mini-implant site and surroundingcortical bone on application of retraction force, at two different insertionangles. post-hoc tests were used to compare thepull-out strength of mini-implants. P values < 0.05 were consideredstatistically significant. Finite element models (FEM) were constructed withinsertion angulation at 90° and 60°, with retraction force of 150 g. Theresults were analyzed using ANSYS software. Mini-implants of three different diameters ,  and  and length of 8mm were used.Using scanning electronic microscopy, the mean thread depth, pitch andrelationship between the two (TSF) were calculated. The mini-implants wereloaded into a synthetic bone block and the pull-out strength was tested. Oneway ANOVA and Tukey Statistically significant difference was found among all the threemini-implants for thread depth and pitch (< 0.001). Statisticallysignificant higher pull-out force value was seen for Orthoimplant. Thestress distribution level in mini-implant and surrounding bone was observedto be smaller for Orthoimplant. Orthoimplant mini-implants have more favorable geometric characteristicsamong the three types, and less stress with 90°angulation. The need for orthodontic treatment modalities that maximize anchorage control andminimize patient compliance has led to the development of mini-implant-assistedorthodontics.-Primary stability of mini-implant is due to the mechanical interlock between the boneand mini-implant, and it depends on many factors, including bone quality,mini-implant site and insertion angle, and design of mini-implants, such asdiameter, thread form, pitch, thread size, mini-implant material,Bone remodeling processes at the bone/screw interface are correlated with thestructural response of the bony tissue to the TADs and then to the stress/strainfield, developing within themselves and the surrounding bone.The proper insertion angle is important for cortical anchorage, patient safety rootdamage), and biomechanical control. It also provides increased surface contact areabetween the mini-implant and the bone.damage, ain vivo is virtually impossible. Thefinite element method (FEM) is thus a valid technique used to analyze structuralstress.Measurement of the stresses en-masse retraction. To address that, this study was conductedto evaluate the effect of TSF of 3 different mini-implants, and their variousinsertion angle combinations, on the pull-out strength and stresses at themini-implant site and surrounding bone during en-masse retraction,using a FEM study.However, the literature lacks information on the combination of ideal geometricdesign characteristics, i.e., TSF and optimal insertion angle duringDetailed geometry of all three mini-implants was studied through scanning electronmicroscope (SEM), to measure the TSF.Pull-out test was carried out to determine the primary stability.FEM was done to evaluate stress distribution at the mini-implant site and in thesurrounding cortical bone, with the application of retraction force at two differentinsertion angles (60° and 90°).The three mini-implants used in the study were as follows:1) ORTHOImplant : 1.8-mm diameter and8-mm length (M1).2) TOMAS (Dentaurum): 1.6-mm diameter and 8-mm length (M2).3) VECTOR TAS (Ormco): 1.4-mm diameter and 8-mm length (M3). According to the manufacturer’s description, these mini-implants are available inthe above mentioned diameter with three different lengths. All mini-implants aremade of Ti-6Al-4V alloy.For the pull-out test, double layer artificial bone block  was used. The block is composedby a polyurethane foam, measuring 120 x 170 x 41 mm thick, having a 1.1-mm toplayer with a 40-pcf density, and a 39-mm base layer with a 10-pcf density .10Each mini-implant was examined using a scanning electron microscope , operating at 30.00 kV, which was performed at Sanraylaboratories Pvt Ltd . Images of each mini-implant werecaptured with VEGA 3.0 software, and obtained at 27× and 33× magnifications. The pitch and thread depth was measured using teammeasurement tool of Biovis Materials VA4.59 software.Pull-out strength tests were performed at the same laboratory on bone blocksconstructed featuring a superficial layer with biomechanical characteristics similar to the cortical bone and a deeperlayer with characteristics mimicking the trabecular bone. Computedtomography study by Migliorati et al.This bone block was divided into small blocks measuring 1.5cm x 2.5cm, sothat it could fit accurately in between the metal plates of the testingmachine. The geometric center was marked on each bone block and themini-implants were inserted at these points, to a thread depth of 6mm. Thepull-out test was carried out by a universal testing machine Shimadzu AGS-Xfeaturing 5 kN load. The mini-implant was loaded with a traction speed of2mm/minute and the pull-out strength was measured as the peak force recordedby the built-in machine software (Trapezium v. 1.4.5). The method wasrepeated for each mini-implant.o and 60o angulations,respectively, is presented in For creating a finite element model, a 3D CAD model was constructed from a CTscan of the craniofacial complex of a 15-years-old female patient. CT scanimages of the maxillary bone were taken by Siemens Somatom Definition 64 in axial direction. Sequential CT images were taken at 0.5mm intervals to reproduce finer and detailed aspects of the geometry. Atotal of 625 images were stacked over one another and converted to a finiteelement meshed model by the software MIMIC (version 18.0). Tetrahydronelements were used to mesh the skull and teeth. Archwire, brackets,crimpable hooks and NiTi closed coil spring were modeled by the softwareANSYS Design Modeler  with beam elements. The total number of elementsin the geometry was 864,650 and the total number of nodes created was247,119 . Nodes and elements defined for each model of mini-implants for 90Only one side of the maxilla was generated, as results on the other side areexpected to be the same. To simulate the extraction space, maxillary firstpremolar was removed from the model. Geometric model of brackets,mini-implants, archwire with crimpable hook and nickel-titanium closed coilspring were constructed using reverse engineering technique. Brackets modelswere constructed using stainless steel MBT prescription of slot size 0.022 x0.028-in. Stainless steel archwire of 0.019 x 0.025-in dimension and anickel-titanium closed coil spring was fabricated and attached fromcrimpable hook to the mini-implant head, generating retraction force of 150g.The minipost-hoc tests were used to compare the TSF andpull-out strength of the mini-implants, within as well as between thegroups. The level of significance was p<0.05. Data wereanalyzed using SPSS software v. 23.0.Descriptive statistics included calculation of mean and standard deviationfor TSF and pull-out tests of three different mini-implants. Shapiro-Wilk’snormality test was used to verify the equality of variance. One-way ANOVAand Tukey Post-hoc Tukey test showed: statistically significantdifference for thread depth between M1 and M2, and M2 and M3; not significantdifference between M1 and M3; and statistically significant difference betweenall the groups for the pitch of the mini-implants .implants .The mean values of M1, M2 and M3 for the pull-out test were 0.181kN; 0.142kN and0.138kN, respectively. Differences were statistically significant . The results showed changes in terms of von Mises stress and principal stresses.The magnitude of stresses developed in reaction to applied retraction force ismentioned in For M1 at 90° insertion angle, maximum stress was observed on the head of themini-implant at the point of attachment with the retraction spring and at thejunction of the head and transmucosal collar (neck). The stresses graduallydecreased from first thread until fourth thread. Minimum levels of stressremained constant throughout the length of the mini-implant , M1.At 60° insertion angle, a small portion of maximum stress was observed at thejunction of the head and neck. The stresses gradually decreased from firstthread until fourth thread. Minimum levels of stress remained constantthroughout the length of the mini-implant. The maximum von Mises stresses at 90°and 60° insertion angle were 23.72 MPa and 29.01 MPa, respectively , M1.For M2 at 90° insertion angle, maximum stress was observed in the first andsecond threads. Stresses decreased towards the neck and below the third thread.The stresses gradually decreased from fourth and fifth thread. The stressesremained minimal from fifth thread to the tip of the mini-implant , M2.At 60° insertion angle, maximum stresses were observed at the larger portion offirst and second threads, and a smaller portion of third thread and neck of themini-implant. Stresses were decreased to a small portion of fourth thread, andthe stresses reached minimum levels and remained constant at the head and alarger portion of neck and from fifth thread to the tip of the mini-implant. Themaximum von Mises stresses at 90° and 60° insertion angle were 80.03 MPa and107.06 MPa, respectively , M2.For M3 at 90° insertion angle, maximum stress was observed at the first andsecond threads and a small portion of the head, at the point of attachment ofretraction spring. The stresses gradually reduced at the neck and a smallportion of third and fourth threads, and from there the stresses reached minimumlevel and remained constant throughout the length of the mini-implant , M3.At 60° insertion angle, maximum stresses were observed at the junction of headand neck. The stresses reduced from the threaded body and at the small portionof third thread. The stresses reached a minimum level and remained constantthroughout the length of the mini-implant. The maximum von Mises stresses at 90°and 60° insertion angle were 17.01 MPa and 14.89 MPa, respectively , M3.For M1 at the 90° and 60° insertion angle, the pattern of stress distribution wasthe same, where maximum stresses were observed at the mesial, distal and apicalto the mini-implant. Stresses uniformly decreased in the form of concentriccircles as it is moved away from the mini-implant and reached closer to theupper small portion of the lower crest of the cortical bone .For M2 and M3 at the 90° insertion angle, the pattern of stress distribution wassimilar to what was observed for the M1. At 60° insertion angle, the stresses atM2 and M3 were close to M1, with a main difference that the stresses reached thebroader area of lower crest of the cortical bone .11-12 and mini-implant designfactors like diameter, pitch, thread depth and TSF.,The fundamental parameter for primary retention of TADs is the pull-out strength,which is linked to bone related factors,The results of this study showed no correlation between TSF and pull-outstrength. Previous literature has shown contradicting conclusions. Radwan etalThe results in the current study showed a non-significant difference in TSF andsignificant difference in pull-out force between the three groups. M1 had thehighest value of pull-out force, followed by M2 and, finally, M3 . The res2reported that there was a positive correlation between pitch and pull-out forcewhen mini-implants of less than 1mm pitch were inserted in cortical thickness of2.2mm. The reason for increased primary stability in that study could be due tomore thread engagement in cortex in mini-implants with < 1 mm pitch whencortical bone was 1.0 - 2.0mm in width.The results of the current study showed no definitive correlation between thepitch and pull-out values, as shown in The current study showed no definitive correlation between the thread depth andpull-out values, as shown in 15 Results of thisstudy were in agreement with results reported in previous studies,,19 OrthoImplantimplants can safely resist the high levels of orthodontic forces used foren-masse teeth retraction and molar uprighting.The greater the diameter of the mini-implant, the greater the bone compressionis, leading to an increased primary stability.The insertion angle of mini-implant varies most often according to clinicalpreference. Therefore, it is necessary to compare the efficacy in terms ofstress induced in the metal and bone among mini-implants of various designand insertion angle with orthodontic loading.In the present study, it was observed that for a given load, i.e. 150g, thestress values on mini-implant and in surrounding bone were higher for M2with 60° and 90° insertion angle, followed by M1 and M3, respectively (Table6). M2 mini-implant, which has a greater thread depth and smaller taperdesign, showed higher stresses when compared to the other two mini-implants.The results of the present study are in agreement with Chang et al,,The stress levels in the mini-implant increased with reduction in theinsertion angle for M2 and M1. The results are in agreement with studies byWoodall et al,24 in their studies suggested that 2-3mmof the implant’s endo-osseous length is most critical in terms of stressresponse under tangential loading, and the results of the present study werein agreement with that. Mini-implants manufacturers should expect morefailures at top three threads.For M2 mini-implant, high stresses were distributed on the uppermost threadsat the neck of the mini-implant near the margin of bone, with both insertionangulations. High stresses were observed on the head of mini-implant at thepoint of attachment of the retraction spring, with respect to M1 and M3 withboth insertion angulations. This pattern of stress distribution on mini-implant (M1 and M3) was in agreement with studies conducted by Ammar etalHowever, the stress values in the current study were below the yield stressof titanium (692 Mpa),-Highest amount of principal stress in the bone were seen with the M2mini-implant and the least amount of principal stress were seen for the M1type. The results were in agreement with previous studies,The maximum principal stress in bone for both insertion angles indicated thatthe stress decreased from 60° to 90° for M1, but this decrease in stressdistribution was observed to be marginal. These findings were in agreementwith previous studies,» Within the limitation of this study involving the finite elementanalyses and mechanical testing of different mini-implants, the resultdemonstrated that Orthoimplant type with a larger diameter, smallerpitch and shorter taper length have better primary stability, and alsohave low stresses within the mini-implants and surrounding bone amongstthe three groups.» The favorable insertion angulation found was 90°, as it provides betterprimary stability and low stresses in the mini-implant and surroundingbone under orthodontic loading. » Further research is required for optimization of thread-parameters andits validation on living bone tissue.